Lates calcarifer). The deduced amino acid sequence of the c-type lysozyme contained 144 residues and possessed typical structure residues, conserved catalytic residues (Glu50 and Asp67) and a “GSTDYGIFQINS” motif. The deduced g-type lysozyme contained 187 residues and possessed a goose egg white lysozyme (GEWL) domain containing three conserved catalytic residues  essential for catalytic activity. Real time quantitative PCR (qRT-PCR) revealed that the two lysozyme genes were constitutively expressed in all the examined tissues. The c-type lysozyme was most abundant in liver, while the g-type lysozyme was predominantly expressed in intestine and weakly expressed in muscle. The c-type and g-type transcripts were up-regulated in the kidney, spleen and liver in response to a challenge with Vibrio harveyi. The up-regulation of the c-type lysozyme was much stronger than that of the g-type lysozyme in kidney and spleen. The recombinant proteins of the c-type and g-type lysozymes showed lytic activities against the bacterial pathogens Vibrio harveyi and Photobacterium damselae in a dosage-dependent manner. We identified single nucleotide polymorphisms (SNPs) in the two lysozyme genes. There were significant associations of these polymorphisms with resistance to the big belly disease. These results suggest that the c- and g-type genes play an important role in resistance to bacterial pathogens in fish. The SNP markers in the two genes associated with the resistance to bacterial pathogens may facilitate the selection of Asian seabass resistant to bacterial diseases.Lysozymes are important proteins of the innate immune system for the defense against bacterial infection. We cloned and analyzed chicken-type (c-type) and goose-type (g-type) lysozymes from Asian seabass ( Solea senegalensis) [Epinephelus coioides) [Larimichthys crocea) [Ctenopharyngodon idellus) [Paralichthys olivaceus) [Scophthalmus maximus) [Oncorhynchus mykiss) [Cyprinus carpio L.) [Escherichia coli show antimicrobial activity against Vibrio anguillarum [Aeromonas hydrophila, Vibrio fluvialis, Aeromonas sobria and Micrococcus lysodeikticus, in gastropod Oncomelania hupensis [V. anguillarum or M lysodeikticus in Mediterranean mussel [V. splendidus and V.parahaemolyticus in Chlamys farreri [Vibrio alginolyticus and Vibrio parahemolyticus in shrimp [Lysozymes are the important antibacterial effector of the innate immunity in animals. They cleave the β-1,4)-glycosidic bond between N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) in the peptidoglycan layer of bacterial cell walls, and ,4-glycos, goose-talensis) , orange-oioides) , large y crocea) , grass cidellus) , Japanesivaceus) , turbot maximus) , rainbow mykiss)  and commrpio L.) . The exprpio L.) ,15. The uillarum  in Japanhupensis , V. angun mussel , V. sple farreri , as welln shrimp . Lates calcarifer Bloch, 1790) belonging to the family Latidae, is known also as Barramundi in Australia. It is widely distributed in tropical Indo-west Pacific, and Northern Australia. Since 1998, we started a breeding program for Asian seabass to improve growth [The Asian seabass . With the two types of recombinant lysozymes, we characterized their lytic activity against V. harveyi, Photobacterium damselae (P. damselae) strains and E. coli DH5α. In addition, we identified SNPs in the two genes and analysed their associations with resistance against the big belly disease. This study could shed new insights on the defence functions of the c-type and g-type lysozymes again bacterial pathogens and supply DNA markers for selection of Asian seabass resistant to the big belly disease.In the present study, in order to investigate the functions of the lysozyme genes against bacterial pathogens we isolated the c-type and g-type lysozyme cDNA and analysed their expression profiles in normal individuals as well as in individuals challenged with All handling of fishes was conducted in accordance with the guidelines on the care and use of animals for scientific purposes set up by the Institutional Animal Care and Use Committee (IACUC) of the Temasek Life Sciences Laboratory, Singapore. The IACUC has specially approved this study within the project “Breeding of Asian seabass” -12-004).Asian seabass individuals were cultured in the Marine Aquaculture Center (MAC), Singapore with the standard operation protocol (SOP) set by MAC. Thirty individuals of healthy Asian seabass at the age of 90 days post-hatch (dph) with an average body weight of 30.05 ± 2.68 g were transported to large tank containing 500 L seawater located in the animal house of our institute three weeks before the commencement of the experiment. The fishes were maintained in the large tank, and were fed twice daily with pelleted feed . http://www.ncbi.nlm.nih.gov/projects/gorf/). The putative signal peptides were analysed using SignalP 3.0 Server . The molecular weight (MW) and theoretical isoelectric point (pI) of the deduced amino acid sequences were analysed using the export protein analysis program (http://au.expasy.org/tools). The full length cDNA for Asian seabass c-type and g-type lysozymes were obtained from cDNA libraries constructed in a previous study ,31. The 2, 0.2 μM of each primer, 50 μM of each dNTP and 1 unit of Taq DNA Polymerase . The PCR program consisted of the following steps: 94°C for 2 min followed by 38 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 40 s, then a final step of 72°C for 5 min. The PCR products were cleaned using NucleoSpin Extract II Column  and cloned into the pGEM-T vector, and then sequenced using M13 and M13R primers and Bigdye chemicals on an ABI 3730xl DNA sequencer . The full-length genomic DNA sequences of the c-type and g-type lysozyme genes were obtained by assembling sequences using software Sequencher 4.9  and submitted to NCBI .To obtain the genomic sequence of the two genes, primers were designed to amplify genomic DNA. The forward primers were located in the 5′ UTR and the reverse primers were in the 3′ UTR , 0.3 μl of each primer (10 pm/μl) and 1 μl  of template cDNA. The cycling conditions consisted of an initial, single cycle of 10 min at 95 °C followed by 40 cycles of 15 s at 95 °C, 30 s at 55 °C and 20 s at 72 °C. After completion of the qPCR, a melting-curve analysis was performed to confirm the specificity of the amplification. qPCRs were performed in triplicates. The expression levels of c-type and g-type lysozymes were analysed using ΔΔCT method [Total RNA was isolated from five fishes at the age of three months using Trizol reagent  according to the manufacturer’s instructions in the following tissues: heart, liver, spleen, gill, kidney, muscle, intestine and brain. RNA was transcribed with MMLV reverse transcriptase  following the supplier’s protocol. The mRNA expressions of the c-type and g-type lysozyme genes of the Asian seabass in the different tissues were detected by real time quantitative PCR (qPCR) with the elongation factor 1-alpha (EF1α) gene as an internal control ,31,36. TT method . V. harveyi, which is a significant pathogen of marine vertebrates and invertebrates. The 30 individuals maintained for three weeks in our fish facility were divided into two groups. Fifteen fishes were transferred into the test tank where each fish was injected intraperitoneally with 0.1 ml of V. harveyi (~e9 copy/ml) in phosphate-buffered saline as described by Xia et al [V. harveyi were compared with the control level at the respective time and tissues. The resulting expression ratio was then converted to natural logs and analysed with Cluster 3.0 (http://bonsai.ims.u-tokyo.ac.jp/~mdehoon/software/cluster/software.htm#ctv).qRT-PCR were conducted to investigate the temporal and spatial expression patterns of the c-type and g-type lysozyme genes in Asian seabass challenged with ia et al . In the ia et al ,38 from 600 of the culture reached 0.6 in liquid LB medium. The cells were cultured at 25°C for 2 h post-induction and the cell suspension was subsequently gently sonicated on ice, and the recombinant lysozyme was released into the supernatant. The supernatant was collected and purified by Glutathione Sepharose 4B affinity chromatography . Half of the fusion proteins were digested with thrombin on the column before elution with PBS, whereas the remaining proteins were eluted from the column with glutathione. Purification and elution were performed according to the instruction manual . The protein was loaded into 12% SDS-PAGE and visualized after staining with Coomassie brilliant blue R-250. The concentration of the purified protein was determined using the Bradford Protein Assay Kit . A forward primer for the c-type and g-type lysozyme genes with an EcoRІ restriction site and a reverse primer with an XhoІ restriction site were designed, respectively, to amplify the coding sequence for the c-type and g-type lysozymes . PCR ampV. harveyi, P. damselae and E.coli DH5α, as V. harveyi and P. damselae are the major pathogens for Asian seabass. The assay was performed according to a modified method by Sun et al [600 of 1.0, collected and diluted using nutrient broth to a density of 2×105 CFU/ml. 12 columns were filled with 0.5 ml nutrient broth contain 2×105 CFU/ml tested strains. 0.5 ml of recombinant proteins was diluted with 0.5 ml nutrient broth in column 1 and blended together. Then serial doubling dilutions in columns 2-10 were conducted. Column 11 served as a growth control, and column 12 was used as blank control. The tubes with V. harveyi and P. damselae were incubated at 30°C for 16 h, and the plates with E.coli were incubated at 37°C for 16 h. Bacterial density was measured according to an absorbance of 630 nm. The 50 mM phosphate buffer saline (pH 7.4) in which the recombinant proteins were dissolved was used as the control.The mature peptides of the c-type and g-type lysozymes were purified with affinity column and released from the fusion protein by digestion with thrombin. In this study, antibacterial testing was carried out using un et al . The bac2 Asian seabass individuals collected from an F2 Asian seabass population. PCR was conducted as described above. The genotyping of four SNPs were conducted by directly sequencing PCR products using an ABI 3730xl sequencer as described above. SNP genotypes were analysed by using the software Sequencher  To identify SNPs in the lysozymes, two pairs of primers  were desIn order to examine whether SNPs in the two lysozymes were associated with resistance to the big belly disease, we genotyped individuals before (as a control group) and after (as a disease-resistant group) an outbreak of the big belly disease in an Asian seabass population generated by a mass cross between 15 males and 15 females. After the outbreak of the disease at the age of the 25 days post hatch (dph), only less than 20% of fish survived at 90 dph. We regarded the surviving individuals after the outbreak of the disease as individuals resistant to the disease. We collected fin clips from 139 and 151 individuals before the outbreak of the disease for genotyping c-type and g-type, respectively, and 175 and 185 individuals at 90 dph (after the outbreak of the disease) for c-type and g-type, respectively. SNPs were genotyped in 314 and 336 fish for c-type and g-type, respectively. The Statistical Program for Social Science (SPSS)  version 12.0 was used for data analysis. The genotype frequency and allele frequency were compared in individuals collected before and after the outbreak of the big belly disease. Chi-square tests were conduct to analyse the significance level of the differences. 50, Asp67), the Ca2+ binding site , and the motif “GSTDYGIFQINS” at positions 64-75 flanking the active Asp67 are also highly conserved among species. The full-length cDNA sequence of the g-type lysozyme of Asian seabass was 710 bp in length and contained an ORF of 564 bp encoding 187 amino acid residues. The 5′- UTR and 3′-UTR of the g-type lysozyme were 12 bp and 134 bp, respectively. Typical signal peptide was not found. The calculated molecular mass of the g-type lysozyme was 20.73 kDa, with a pI of 7.99. In silico analyses revealed that the g-type lysozymes possesses a Goose Egg White Lysozyme (GEWL) domain (residues 16 to 185) containing three conserved catalytic residues Glu71, Asp84 and Asp95. Multiple sequence alignment of the g-type lysozyme with other fish counterparts .The full-length cDNA sequence of c-type lysozyme was 566 bp and contained an open reading frame (ORF) of 435 bp. The 5′-untranslated region (UTR) and 3′-UTR of the c-type lysozyme cDNA sequence were 27 bp and 104 bp, respectively. The c-type lysozyme was predicted to encode a polypeptide of 144 amino acid (aa) residues with the signal peptide comprising the first 15 residues . The matterparts  revealedSolea senegalensis and rainbow trout, Oncorhynchus mykiss. The g-type lysozyme of Asian seabass was located in the same branch as the Mandarin fish, Siniperca chuatsi, and also closely related to the g-type lysozyme of European seabass, Dicentrarchus labrax and the Senegalese sole.A phylogenetic tree  was consThe genomic sequence of the c-type lysozyme gene from the transcriptional start site to the transcriptional end site consisted of 1491 bp, comprising of four exons  and three introns (Accession no KC460318). The genomic DNA sequence of the g-type lysozyme gene consisted of 1232 bp, comprising of five exons  and four introns (Accession no. KF183642).p < 0.05).Expressions of the c-type and g-type lysozymes were detected in all tested tissues of untreated individuals . The higV. harveyi were measured by qRT-PCR , which was the highest expression level among the described tissues. As time progressed, the expression of the c-type lysozyme in the challenge group dropped back to the normal level, without significant difference compared with the control. The gene expression showed similar profiles in liver, kidney and spleen. The expression levels of g-type lysozyme were up-regulated drastically and then decreased to the normal level. The peak value appeared at 3 h with 3.2-fold increase in kidney, 6 h with 2.5-fold in spleen, and 12 h with 1.5-fold in liver (p < 0.05). These data indicate that c-type and g-type lysozyme gene may contribute to the innate immune defence upon bacterial infection.In order to investigate a potential immunological function and the response to bacteria for both lysozyme types, the temporal expressions of the c-type and g-type lysozymes in liver, spleen and kidney of the Asian seabass challenged with  qRT-PCR . ControlE. coli BL21 (DE3) as described above. After IPTG induction, the whole cell lysate was analysed by SDS-PAGE. The lysozyme protein gave a distinct band at about 36 kDa (GST-protein 20 kDa + c-type mature peptides 16 kDa) for c-type lysozyme and 40.13 kDa (GST-protein 20 kDa + g-type mature peptides 20.13 kDa) for g-type lysozyme as compared with the control  of the c-type and g-type lysozymes could inhibit the growth of t manner . Meanwhip < 0.05) between individuals before and after the outbreak of the big belly disease. The four SNP alleles C, T, A and G at the four SNP loci  was located in exon 2, and the other  was in intron 3. In the g-type lysozyme, two SNPs were located in exon 1 . SNPc1 in c-type lysozyme and SNPg1, SNPg2 in g-type lysozyme were found in exon sequences, and resulted in amino acid changes. The allele and genotype frequencies of the four c-type and g-type SNPs are shown in SNP loci  were freWe identified two lysozyme genes from the Asian seabass. A phylogenetic analysis of the amino acid sequences revealed that the two genes were the c-type and g-type lysozyme genes from Asian seabass. We further analyzed their molecular characteristics, expression and regulation after bacterial infections. The predominant molecular characteristics of c-type and g-type are as follows. Firstly, the c-type lysozyme in Asian seabass, containing the deduced 143 amino acid residues, is similar to other fish species, such as, rainbow trout , Nile ti71, Asp84 and Asp95), except the common carp in which the second catalytic residue Asp86 was replaced by proline [O. hupensis [In fish and mammals, the presence of two putative conserved catalytic residues (Glu and Asp) in c-type lysozyme are critical for the lysozyme lytic activity to bacterial cell wall . The c-t proline . G-type  proline . Howeverhupensis . Thus, tWe examined the expressions of the c-type and g-type lysozymes in different tissues. The c-type lysozyme was highly expressed in liver, intestine, spleen and kidney, but expression was low in other analyzed tissues. Similar expression of the c-type lysozyme was seen in zebrafish  and rainV. harveyi challenge was recorded to further investigate their functions. The results indicate that the c-type and g-type lysozyme genes were up-regulated in some tissues when challenged with the pathogen, V. harveyi. The strongest up-regulation of c-type lysozyme mRNA levels was detected in both kidney and spleen  was associated with susceptibility/resistance to Listonella anguillarum [Meretrix meretrix, SNPs in the i-type lysozyme gene was associated with Vibrio resistance [Four SNPs were detected in these two lysozyme genes of Asian seabass. Analysis of allele frequencies in the control and the big belly resistant groups revealed significant differences of allele frequencies, suggesting that the SNPs may be associated with the resistance to big belly disease in Asian seabass. In other species, polymorphisms in lysozyme genes were also found to be associated with resistance to bacterial diseases ,53. For sistance . The fousistance .V. harveyi and P. damselae suggest that these two lysozymes are important in the defense against invading bacterial pathogens. The purified recombinant proteins of these two lysozyme genes show obvious antibacterial activities. We also showed significant associations (p < 0.05) between lysozyme polymorphisms and resistance against the big belly disease. This study provides new insights on the antibacterial functions of the c-type and g-type lysozymes. The four SNPs in the two genes associated with the resistance to bacterial pathogens may facilitate the selection of Asian seabass resistant to the big belly disease. In conclusion, the c-type and g-type lysozymes were identified and characterized in the Asian seabass. The expression levels in various tissues after being challenged with Figure S1Alignment of amino acid sequence of lysozyme homologs.Left side: “*” indicates the identical amino acid residues; (: or .) are indicates similar residues. Lysozyme catalytic residues Glu (E) and Asp (D) are bold; lysozyme catalytic cleft is gray with underline; Ca2+ binding site is boxed; N-acetyl-D-glucosamine binding site is gray; conserved motif is black shading. c-type lysozymes: Human (Homo sapiens), AAI00886; Mouse (Mus musculus), NP_059068; Pig (Sus scrofa), AAB16862; Rainbow trout (Oncorhynchus mykiss), AF321519_1; Medaka (Oryzias latipes), ACO82287; Nile tilapia (Oreochromis niloticus), XP_003457546; Fugu rubripes (Takifugu rubripes), NP_001027914.1; Yellow perch (Perca flavescens), ACO34809; Senegalese sole , ABC49680; Asia seabass , KF183643. 2+ binding site is boxed; N-acetyl-D-glucosamine binding site is gray; conserved motif is black shading. g-type lysozymes: Human (Homo sapiens), AAI00886; Mouse (Mus musculus), AAI47568.1; Asia seabass , ABV66069; European seabass (Dicentrarchus labrax), CBJ56263; Rainbow trout (Oncorhynchus mykiss), ACO08589; Blue catfish , ADO28271; Gilt-head bream (Sparus aurata), CAO78618; Fugu rubripes (Takifugu rubripes), NP_001027764; Senegalese sole , BAG14278; Mandarin fish (Siniperca chuatsi), AAU86896.Right side: “*” indicates the identical amino acid residues; (: or .) indicates similar residues. Lysozyme catalytic residues Glu (E) and Asp (D) are bold; lysozyme catalytic cleft is gray with underline; Ca(TIF)Click here for additional data file.Figure S2A NJ-phylogenetic tree of c-type and g-type lysozyme genes.(TIF)Click here for additional data file.


In vitro studies using the MG-63 cell line revealed profound cytocompatibility, increased cell proliferation and enhanced alkaline phosphatase secretion in the Chi-Alg-fucoidan scaffold compared to the Chi-Alg scaffold. Further, protein adsorption and mineralization were about two times greater in the Chi-Alg-fucoidan scaffold than the Chi-Alg scaffold. Hence, we suggest that Chi-Alg-fucoidan will be a promising biomaterial for bone tissue regeneration.Over the last few years, significant research has been conducted in the construction of artificial bone scaffolds. In the present study, different types of polymer scaffolds, such as chitosan-alginate (Chi-Alg) and chitosan-alginate with fucoidan (Chi-Alg-fucoidan), were developed by a freeze-drying method, and each was characterized as a bone graft substitute. The porosity, water uptake and retention ability of the prepared scaffolds showed similar efficacy. The pore size of the Chi-Alg and Chi-Alg-fucoidan scaffolds were measured from scanning electron microscopy and found to be 62–490 and 56–437 µm, respectively.  Bone, a complex and hierarchical tissue with a major portion made up of hydroxyapatite (HA) and collagen, plays a major role in the structural framework, mineral deposition, pH regulation and mechanical support. Bone defects or damages are possible in a number of ways, including motor accidents, birth defects and chronic diseases. Over 2.2 million-bone graft procedures are performed annually worldwide ,2. Severd-glucosamine and N-acetyl glucosamine linked in a β (1-4) manner. Chitosan possesses excellent biocompatibility, biodegradation, antimicrobial activity and low immunogenicity. It can be molded into various forms  and has an exceptional pore forming ability for potential applications in tissue engineering, drug delivery and wound healing [d-mannuronate and α-l-guluronate residues. These homopolymeric blocks are covalently linked together in sequences that include blocks of alternating α-l-guluronate-β-d-mannuronate copolymer. Alginate is commonly isolated from marine seaweed. Similar to chitosan, alginate is also an exceptional biomaterial for bone tissue engineering, due to its biocompatibility, biodegradability, non-antigenicity, encapsulation capacity, chelating ability and ability to be cast in different forms, such as gels, microspheres, foams, fibers and sponges. Alginate scaffolds are often used for delivering materials, such as bone morphogenetic protein-2 (BMP-2) and mesenchymal stem cells (MSC) to the defective area for repairing the tissues [N-succinyl-chitosan for bone tissue regeneration [Chitin is a natural polysaccharide, and it is the most important polymer after cellulose. It occurs in the exoskeleton of arthropods or in the cells walls of fungi and yeast. An important derivative of chitin is chitosan. It is obtained by partial deacetylation of chitin using a chemical method (concentrated NaOH) or by enzymatic hydrolysis . Chitosain a β 1- manner.  healing . Chitosa healing ,11,12,13 tissues ,15. Chi- tissues ,20,21,22 tissues ,24,25, t tissues  and inte tissues . In factneration . Apart fneration .l-fucose and sulfate. It is commonly found in marine brown seaweeds. Fucoidan can increase the level of alkaline phosphatase (ALP), type-1 collagen expression, osteocalcin and BMP-2 and even helps in mineral deposition associated with bone mineralization [in vitro and in vivo [Fucoidan is a sulfated polysaccharide that contains lization ,31. In alization . Composilization ,34. Arou in vivo .Hence, considering the biocompatibility, biodegradation, antibacterial nature, film-forming ability and induction of osteogenic differentiation by fucoidan, Chi and Alg, we aimed to report the synthesis and characterization of the newly developed Chi-Alg biocomposite containing fucoidan for bone tissue engineering.In this study, scaffolds were fabricated by a freeze-drying method. The Chi-Alg and Chi-Alg-fucoidan scaffolds were found to be stiff and inelastic. Chi-Alg scaffolds were observed in a colorless state, whereas Chi-Alg-fucoidan scaffolds obtained a pale brown color, which is due to the dispersion of fucoidan in the composites. By the visual observation , fucoidaAlginate is an anionic polymer that possesses the ability to form strong electrostatic interaction with cationic polymers. In this case, the cationic charged amine group of the chitosan unit interacted electrostatically with the negatively charged alginate to form a polyelectrolyte mixture. The addition of fucoidan in the Chi-Alg may make more complex ionic interactions possible. Several studies suggested that hydrogen bonding or ion-ion pair interaction between the components usually increases the uniform dispersion . These m−1, which corresponds to the carboxylate group (C=O). In addition, a strong intense peak was observed at 3446 cm−1, corresponding to the –OH group. The symmetric stretching frequency of the carboxyl group was observed at 1418 cm−1, whereas 1098–1026 cm−1 shows the asymmetric stretching frequency. Chitosan shows vibration at 3433 cm−1 (–OH and N-H stretching vibrations), 2855 cm−1 (C-H stretch), 1640 cm−1 (amide I), 1575 cm−1 (N-H bending of amine) and 1029 cm−1 .The alginate spectrum shows the characteristic peak at 1623 cm−1, corresponding to the superposition of the bands assigned to the carboxylate group of alginate and the amine group of chitosan. The interaction from electrostatic interaction between the carboxylate group of alginate and the amine group of chitosan forms a polyelectrolyte complex. The results are consistent with previous studies [−1 to 3420 cm−1. This suggests that intermolecular hydrogen bonds exist in the chitosan-alginate system [On the other hand, in the case of Chi-Alg, an intense peak was observed at 1613 cm studies . The lowe system .−1 assigned to the (S=O) group and a sharp band at 842 cm−1 assigned to the sulfate group in fucoidan [−1 corresponded to the –OH group on the fucoidan moiety. The corresponding stretching frequency of fucoidan is not clearly visible in the Chi-Alg-fucoidan composite. This may be due to the fact that the level of fucoidan in the Chi-Alg-fucoidan scaffold was too small to be detected.Fucoidan exhibits its characteristic bands at 1210–1280 cmfucoidan . The peaThe porosity of the prepared scaffolds was measured through the liquid displacement method using ethanol. The results suggest that the porosity of the scaffolds are >90%. The porosity of the Chi-Alg and Chi-Alg-fucoidan scaffolds were measured as 94.5% ± 0.5% and 94.9% ± 0.2%, respectively. Greater than 90% total porosity was observed for the polymeric scaffold, which could be an added advantage for tissue engineering purposes . This hiThe water uptake ability of the scaffolds is measured by the swelling behavior of the scaffold in phosphate buffer saline (PBS) solution. The water uptake and retention ability of the Chi-Alg and Chi-Alg-fucoidan were studied by immersing these scaffolds in 1× PBS solution a. The rein vivo study. Proteins, including fibronectin, vitronectin and other signaling molecules, can be adsorbed by the scaffolds from the circulating body fluids, which facilitate cell adhesion, proliferation and differentiation. The amount of adsorbed protein on the Chi-Alg and Chi-Alg-fucoidan scaffolds was measured with respect to time. The scaffolds were incubated with Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% Fetal Bovine Serum (FBS), and as shown in The study of the protein adsorption of the prepared scaffold plays a significant role in In vitro biodegradation is a crucial parameter to be considered in bone tissue engineering. The biodegradation of scaffolds provides space for tissue growth and matrix deposition. In the present study, no difference was observed in the biodegradation in Chi-Alg (15%) and Chi-Alg-fucoidan (15%) at 24 h. However, higher degradation was observed in the Chi-Alg-fucoidan scaffold (40%) compared to Chi-Alg (15.7%) at 72 h. This might be because of the electrostatic interactions between chitosan and fucoidan, as well as the weak ionic bonding forces between fucoidan and calcium in PBS.The surface morphology, pore distribution and pore size of Chi-Alg and Chi-Alg-fucoidan were examined using scanning electron microscopy analysis . The porin vivo study. An ample number of assays are available to measure the cytotoxicity of the scaffolds, such as the MTT -2,5-diphenyl tetrazolium), WSTs  and LDH (Lactate dehydrogenase) assays. In the present study, we have used the MTT assay to measure the biocompatibility of the prepared scaffolds. This assay is based on the ability of cellular mitochondrial dehydrogenase to reduce the yellow-colored tetrazolium salt to blue-colored formazan crystals. Human osteoblast-like cells (MG-63) were used in the experiment to measure the toxicity level of the prepared scaffolds. Cell viability on the fabricated scaffolds (Chi-Alg and Chi-Alg-fucoidan) at different time intervals is shown in The toxicity and biocompatibility of the prepared scaffolds are important concerns before proceeding to the et al. (2009) reported that Undaria pinnatifida-derived fucoidan significantly induced the osteoblastic differentiation required for bone formation, by increasing the activity of the phenotypic markers, alkaline phosphatase and osteocalcin [The measured ALP activity of the scaffolds are shown in eocalcin . The undin vivo [The low molecular weight of fucoidan suggested that it has the capacity to promote osteoblast proliferation, increase fibrillar collagen content and induce mineralization, which is essential for bone tissue growth . One hunin vivo .2) and frozen at −24 °C for 1 day and freeze dried to form scaffolds. These scaffolds were immersed or cross-linked with 10% CaCl2 solution for 30 min, followed by soaking in absolute ethyl alcohol for 10 min. Finally, scaffolds were washed with an excess amount of water and freeze dried again for experimentation. In the present study, we have used Chi and Alg in a 3:1 weight ratio for the Chi-Alg scaffold fabrication.Alginate (3% w/v) was dissolved in 100 mL of water by using a mechanical stirrer  for 1 h to make a homogeneous solution. Secondly, chitosan  was dissolved in 50 mL of 2% acetic acid solution and was carefully added into the alginate solution, with the help of a dropper. The homogeneous gel solution was stirred at 500 rpm for 1–2 h at room temperature. This gel solution was transferred into the tissue culture dish  was dissolved in 50 mL of 2% acetic acid solution and was carefully added into the alginate-fucoidan solution, with the help of a dropper. This chitosan-alginate-fucoidan gel solution was transferred into the tissue culture dish, and the rest of the procedure was followed as described for the chitosan-alginate scaffold preparation. Ratios of Chi, Alg and Fucoidan of 3:1:0.1 weight were used in the construction of the Chi-Alg-fucoidan system.−1 to 400 cm−1.FT-IR was used to characterize and to know the chemical interactions between the Chi-Alg and Chi-Alg-fucoidan scaffolds. The spectra of the chitosan, alginate, fucoidan, Chi-Alg and Chi-Alg-fucoidan composite scaffolds were recorded using the KBr pellet method in an FT-IR spectrophotometer  with the range of 4000 cm1 = the initial known weight of the scaffold, V2 = the sum of the weights of ethanol and the submerged scaffold and V3 = the weight of ethanol after the removal of the scaffold.The total porosity was determined by the liquid displacement method. Initially, the volume of the ethanol and the dry weight of the scaffolds were measured. Then, the scaffold was immersed into the dehydrated alcohol for 48 h until absorbing the alcohol saturated it, and the scaffold was weighed again. Finally, the porosity of the sample was calculated based on the following formula:Three parallel sets were analyzed for every scaffold, and the mean value of the porosities of different scaffolds was achieved.dry) and immersed in 1× PBS solution for 24 h. Then, the scaffolds were gently removed from the beaker after 24 h and placed on a wire mesh rack. Excessive water was drained, and the scaffolds were weighed (Wwet) after 5 min to determine the water uptake. To measure the water retention ability, the wet scaffolds were transferred to centrifuge tubes containing filter paper at the bottom, centrifuged (Combi 514-Hanil Science) at 500 rpm for 3 min and weighed immediately (W'wet). The percentages of water absorption (EA) and water retention (ER) of the scaffolds at equilibrium were calculated using the following equations:The water uptake and retention ability of scaffold were studied, as described in our previous study ,42. Dry The protein adsorption ability of the Chi-Alg and Chi-Alg-fucoidan scaffolds was measured, as per a previous report . BrieflyI, followed by immersion in 1× PBS containing lysozyme (1000 U/L) and incubated at 37 °C at different intervals (24 and 72 h). After completion of the incubation period, the scaffolds were washed with deionized water to remove ions and blot dried with filter paper. The dry weights of the scaffolds were noted as (Wt). The degradation was calculated by using the following formula:The rate of degradation of the biocomposite scaffolds was studied according to the previous method . The ChiThe pore size and surface morphology of the biocomposite scaffolds were studied using scanning electron microscopy . Briefly, scaffold samples were cut into small pieces and fixed on carbon tape, then dried under vacuum and gold coated before examining under SEM. In addition, optical microscopy was also performed with the prepared scaffolds using an optical microscope .2 and 95% air. The MTT assay was used to measure the cytotoxicity of the prepared scaffolds. Osteosarcoma MG-63 cells with the concentration of 1 × 105 cells/100 μL were seeded dropwise on the small pieces of scaffold and incubated at 37 °C. The cell culture media was removed on the respective days and incubated with fresh medium containing 200 μL of MTT -2,5-diphenyl tetrazolium) for 4 h in darkness. After the incubation period, the MTT dye was removed, followed by the addition of 200 μL DMSO to solubilize the formazan crystals, and optical densities (OD) were determined at 570 nm using a spectrophotometer .The scaffolds (Chi-Alg and Chi-Alg-fucoidan) were cut into small pieces and placed in 24-well plates and incubated with cell culture media (DMEM) for 4 h at 37 °C in an incubator with 5% CO4 cells/mL of MG-63 cells were seeded on the plate and kept for 7 days in the incubator. The ODM was replaced every 2 days. After the incubation, cells were rinsed with PBS, and 100 µL of 25 mM carbonate buffer (pH 10.3) containing 0.2% Tritox X-100 were added. Each well of the plate was transferred with 50 µL of 250 mM carbonate buffer (pH 10.3) containing 2.5 mM MgCl2 and 15 mM p-nitro phenyl phosphate (p-NPP). The plate was incubated for 30 min at 37 °C, and the absorbance was measured at 405 nm in a spectrophotometer .For ALP activity, scaffolds were immersed in 500 µL of osteogenic differentiation medium (ODM) in 24-well culture plate for 3 h, and 5 × 103 cells/100 µL). The media were changed every 2 days until 14 days, then the media were removed. The cells were washed twice by PBS and fixed in 70% ethyl alcohol for 1 h at room temperature. The ethyl alcohol-fixed cells and scaffolds were stained with 40 mM alizarin red S (pH 4.1) for 10 min. Cells were washed with deionized water five times and with PBS two times, then the cells were incubated in 10 mM of sodium phosphate buffer (pH 7.0) containing 10% of cetylpyridium chloride for 15 min. The optical density was measured at 562 nm using a GENios R microplate reader .The mineralization assay was performed by alizarin red S stain. Briefly, scaffolds were immersed in ODM in a 24-well plate for 3 h. After 3 h, MG-63 cells were seeded dropwise into the plate (5 × 10All the data are expressed as means ± the standard deviation of a minimum of three replicates for each scaffold in each experiment, using Graphpad Prism 5.0.In this study, we prepared two different types of composite scaffolds. Chemical ionic interactions were observed between chitosan, alginate and fucoidan, which led to the improved bioactivity of the scaffolds. The addition of negatively charged and sulfated fucoidan in Chi-Alg showed a better activity towards bone tissue regeneration. Owing to the great water uptake ability, sufficient porosity, enhanced protein adsorption and increased mineralization effects, the Chi-Alg-fucoidan scaffolds would be promising biomaterials for bone tissue engineering.


N-terminal prohormone of brain natriuretic peptide (NT-proBNP) and dynamics of other biomarkers related to left ventricular remodeling, fibrosis and angiogenesis.The purpose of the Occluded Artery Trial (OAT) Biomarker substudy was to evaluate the impact of infarct related artery (IRA) revascularization on serial levels of Patients were eligible for OAT-Biomarker based on the main OAT criteria. Of 70 patients  enrolled in the substudy, 37 were randomized to percutaneous coronary intervention (PCI) and 33 to optimal medical therapy alone. Baseline serum samples were obtained prior to OAT randomization with follow up samples taken at one year. The primary outcome was percent change of NT-proBNP from baseline to 1 year. The secondary outcomes were respective changes of matrix metalloproteinases (MMP) 2 and 9, tissue inhibitor of matrix metalloproteinase 2 (TIMP-2), Vascular Endothelial Growth Factor (VEGF), and Galectin-3.Paired (baseline and one-year) serum samples were obtained in 62 subjects. Baseline median NT-proBNP level was 944.8  ng/L and decreased by 69% during follow-up (p < 0.0001). Baseline MMP-2 and TIMP-2 levels increased significantly from baseline to follow-up , while MMP-9 level decreased from baseline (p = 0.038). Levels of VEGF and Galectin-3 remained stable at one year (p = NS for both). No impact of IRA revascularization on any biomarker dynamics were noted.There were significant changes in measured biomarkers related to LV remodeling, stress, and fibrosis following MI between 0 and 12 month. Establishing infarct vessel patency utilizing stenting 24 hours-28 days post MI did not however influence the biomarkers’ release. Myocardial infarction (MI) initiates healing and repair responses, which include scar formation, angiogenesis and remodeling. Preliminary data suggested that late beyond 24 hours) opening of occluded culprit infarct artery might have beneficial impact upon these processes, with subsequent clinical advantages . The Occ4 hours oN-terminal prohormone of brain natriuretic peptide (NT-proBNP) obtained at baseline and at 1 year of follow up; and secondly to assess the impact of the randomized treatment on dynamics of biomarkers related to post-infarction left ventricular remodeling, fibrosis and angiogenesis, including matrix metalloproteinases (MMP) 2 and 9 and tissue inhibitor of matrix metalloproteinase 2 (TIMP-2), Vascular Endothelial Growth Factor (VEGF), and galectin-3.The primary purpose of the OAT Biomarker substudy was to evaluate the impact of mechanical revascularization on paired levels of plus on-year) blood samples were available.The patients were eligible for OAT-Biomarker ancillary study when they consented for the parent trial OAT . Due to lack of funding the study was performed in only 10 sites in Poland, two in Canada and a solitary site in the United States. The substudy was approved by Institutional Review Board at each participating center  and patients provided separate written informed consent for the OAT-Biomarker ancillary study. During follow-up repeated blood sampling 1 year after randomization was performed. For ninety percent (62/70) of the patients paired  for this batched analysis were determined using an Elecsys 2010 . The analytic range of NT-proBNP assay extends from 5 to 35000 ng/L. The total coefficient of variation (CV) was 2.1% at a level of 181 ng/L and 1.8% at a level of 572 ng/L. Concentrations of matrix metalloproteinases, tissue inhibitor of metalloproteinase-2 and VEGF were assayed using quantitative sandwich enzyme immunoassay technique. The following Quantikine  tests were used: human/mouse MMP-2, total , human MMP-9, total, , human TIMP-2 , human VEGF . Concentration of galectin was estimated using human galectin-3 ELISA kit  .The primary endpoint for this biomarker analysis was NT-proBNP percent change from baseline to 1 year. The dynamics of the other biomarkers comprised the secondary outcomes of the study. The primary clinical endpoint of OAT was a composite of death from any cause, reinfarction, or NYHA class IV heart failure with hospitalization or admission in a short-stay unit with follow up extending out to 7 years. The study clinical endpoints were adjudicated by an independent committee. The definition of reinfarction has been provided previously .Data management and statistical analyses were performed by the OAT data coordinating center. Baseline biomarkers were assessed as medians (interquartile range), clinical characteristics were assessed as frequencies/proportions or mean ± standard deviation (SD) and compared to the remaining main OAT patients, as well as by treatment groups (PCI vs. MED). The nonparametric median Wilcoxon test, t-test or chi square tests were used for comparisons as appropriate. The biomarker’s levels were presented as medians (inter-quartile range), and compared by means of Wilcoxon Signed Rank test. Two-sided two-group t-tests at alpha = 0.05 were done for comparisons of biomarkers changes from baseline to 1-year.versus follow-up), biomarkers dynamics between PCI versus MED subgroups, and for regression models with NT-proBNP dynamics as the dependent variable, significance level was set at p < 0.05. For other analyses significance level was set at p < 0.01. All analyses are performed on an intention-to-treat basis.To examine the impact of the study treatment on biomarker dynamics, change in biomarkers’ levels between baseline and 1 year were compared by treatment assignment (PCI versus MED). The independent correlates of NT-proBNP and other biomarkers dynamics (change from baseline to one year levels) were assessed by means of regression analyses. The univariate models were run first to identify any predictor significant at 0.10 level. Then a backward selection was run on the multivariate model to determine the final predictors that were still significant at 0.05 level. The treatment group was forced into the multivariable models. For the biomarkers dynamics , there was 80% power to detect a difference of 56% for the primary outcome of change in NT-proBNP levels from baseline to 1 year.Patient characteristics at study baseline and comparison to the main OAT cohort are illustrated in Table Baseline levels of NT-proBNP were 944.8 455.3, 1533) ng/L and decreased by 69% to 266.8  ng/L during follow-up (p < 0.0001). During this period MMP-2  and TIMP-2 levels  increased significantly from baseline to follow-up. In contrast, MMP-9 levels decreased from 101.6  ng/mL to 80.2  ng/mL (p = 0.038) while levels of VEGF [39.9  pg/mL vs. 48.3  p = 0.291], and Galectin  remained stable  performed 3 to 28 days post MI.NT-proBNP is an established biomarker used for diagnosis, prognosis and in selected cases management of patients with heart failure or acute coronary syndromes. It is synthesized in the ventricular myocardium in response to increased wall tension, myocardial stretch or ischemia -6. The kMatrix metalloproteinases (MMP) and their tissue inhibitors (TIMPs) are enzymes involved in the extracellular matrix turnover and cardiac remodeling following infarction. Their release and roles following MI are time dependent ,14. PrioIn our cohort the values of MMP-2 significantly increased, while MMP-9 decreased from baseline. Similar shifts in these biomarker levels during long-term follow-up were previously observed in patients after MI ,18,21,22TIMP-2 may either inhibit or stimulate activation of MMP-2 ,24. Its VEGF is implicated in neovascularization of ischemic tissue and wound healing, and its levels were previously shown to be correlated with peak creatine kinase levels in patients with MI. VEGF elevation has also been shown to be associated with clinical myocardial ischemia and adverse outcomes following ACS ,27. The Experimental observations suggest that galectin-3 may be regarded not only as a biomarker but also as an active mediator of fibrosis causing progression of heart failure ,29. In tThe study cohort represents a small series of patients enrolled in the main OAT study. The power to detect small differences in the biomarker dynamics between the PCI and MED groups was limited due to the insufficient recruitment relative to initial projections. It is also conceivable that the differences between the study groups would differ had the patients been followed for a longer period of time. Only paired samples at baseline and 1 year of follow up were available. As a result the time course of changes during this window could not be elicited.The Biomarker ancillary study to OAT suggests that there are significant dynamic changes (from baseline to one year) in biomarkers related to LV remodeling, stress, and fibrosis following MI, however, their dynamics are not significantly modified by opening of occluded IRA 3 days-28 days post MI. Consistent with the findings of the parent trial and the nuclear substudy, our findings appear to also suggest that late IRA recanalization does not significantly impact mechanisms underlying LV healing/remodeling processes following MI in patients on optimal medical therapy.The authors declare that they have no competing interests.MK – 1) have made substantial contributions to conception and design, acquisition of data, analysis and interpretation of data; 2) have been involved in drafting the manuscript; and 3) have given final approval of the version to be published; VM, ZS, WR, JH, GO, KZ, SF, DL have made substantial contributions to conception and design, analysis and interpretation of data; 2) have been involved in revising the manuscript critically for important intellectual content; and 3) have given final approval of the version to be published; JK, JJ, MR, PC, MK, BBP, EZ, WJ, KJ, AR, GS 1) have made substantial contributions to acquisition of data; 2) have been involved in revising the manuscript critically for important intellectual content; and 3) have given final approval of the version to be published.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2261/13/91/prepub


VGAT mRNAs and proteins were specifically absent in the 40-week-old L7-VGAT PCs. The morphological characteristics, such as lamination and foliation of the cerebellar cortex, of the L7-VGAT mice were similar to those of the control littermate mice. Moreover, the protein expression levels and patterns of pre-  and postsynaptic (GABA-A receptor α1 subunit and gephyrin) molecules between the L7-VGAT and control mice were similar in the deep cerebellar nuclei that receive PC projections. However, the L7-VGAT mice performed poorly in the accelerating rotarod test and displayed ataxic gait in the footprint test. The L7-VGAT mice also exhibited severer ataxia as VGAT deficits progressed. These results suggest that VGAT in cerebellar PCs is not essential for the rough maintenance of cerebellar structure, but does play an important role in motor coordination. The L7-VGAT mice are a novel model of ataxia without PC degeneration, and would also be useful for studying the role of PCs in cognition and emotion.γ-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the adult mammalian central nervous system and plays modulatory roles in neural development. The vesicular GABA transporter (VGAT) is an essential molecule for GABAergic neurotransmission due to its role in vesicular GABA release. Cerebellar Purkinje cells (PCs) are GABAergic projection neurons that are indispensable for cerebellar function. To elucidate the significance of VGAT in cerebellar PCs, we generated and characterized PC-specific VGAT knockout (L7-VGAT) mice.  The actThe cerebellum is important for motor function, cognition, and emotion . CerebelHere, to study the roles of VGAT in PCs, we generated cerebellar PC-specific VGAT KO (L7-VGAT) mice and examined the cerebellar structure and motor function of these mice, as indices of the developmental failure and dysfunction of adult PCs, respectively. In these knockout mice, the overall cerebellar structure was not altered, but motor discoordination was observed. These results indicate that VGAT in cerebellar PCs is critical for motor coordination in animals and the L7-VGAT mice are a novel mouse model of cerebellar dysfunction without apparent structural alterations in the cerebellum.VGATfloxneo/+ mice) in which the 5′-loxP site was introduced into the XbaI site in intron 1 and an frt-flanked PGK-neo cassette followed by the 3′-loxP sequence was inserted into the KpnI site in the 3′-flanking region (VGATfloxneo/+ mice were mated to FLP66 transgenic mice (VGATflox/+ mice were intercrossed to generate VGATflox/flox mice in which exons 2 and 3 of the VGAT gene were flanked by loxP sites. The generation of L7-Cre knock-in mice has been previously described (L7/Pcp-2 (L7) gene, and the Cre recombinase is expressed under the control of the endogenous L7 promoter. VGATflox/flox mice were crossed with heterozygous L7-Cre knock-in mice (L7Cre/+ mice) to obtain VGATflox/flox; L7Cre/+ mice. VGATflox/flox; L7Cre/+ mice were mated to VGATflox/flox; L7+/+ mice to obtain VGATflox/flox; L7Cre/+ mice and VGATflox/flox; L7+/+ mice, which were used in the subsequent experiments. We refer to the VGATflox/flox; L7Cre/+ mice and the VGATflox/flox; L7+/+ mice as L7-VGAT mice and control mice, respectively, hereafter.We generated heterozygous mice carrying one floxneo allele . Every effort was made to minimize the number of animals used and their suffering.VGAT+/+, VGATflox/+, and VGATflox/flox mice were determined by PCR using the following oligonucleotides: primer 1 (5′-TCAGAGGCTTCTTCCTAGGGCTGCTG-3′) and primer 2 (5′-GACCTCCCCCATTGCATAGAATGGCAC-3′), which correspond to the 3′-flanking region of the VGAT gene. Primers 1 and 2 amplified a 183-bp fragment specific for the wild-type allele and a 310-bp fragment specific for the floxed allele. The following primers were used for the L7+/+, L7Cre/+, and L7Cre/Cre genotype mice: primer 3 (5′-CCAGGAAGGCTTCTTCAACCTGCT-3′), primer 4 (5′-CCTGGGTGTTGACCAGCATATCC-3′), and primer 5 (5′-GTACGGTCAGTAAATTGGACATGCGG-3′). Primers 3 and 4 amplified a 311-bp fragment specific for the wild-type allele, and primers 3 and 5 yielded a 152-bp fragment specific for the Cre knock-in allele  were removed and embedded in Tissue-Tek OCT compound . Samples were sectioned on a cryostat  at 10 μm. The cryosections were mounted onto SuperFrost Plus microscope slides , air-dried for more than 20 min at room temperature, and stored at –80°C until use. The thicknesses of the granule cell and molecular layers and the densities of the PC somata were determined using sections stained with cresyl violet, and two sections were prepared from each animal. Laminar thickness was measured at five points along the rostral aspect of lobules IV–V in each section. The densities of the PC somata were determined by dividing the total number of PCs per section in lobule III by the length of the entire PC layer in this lobule. PCs were defined as cells with diameters greater than 15 μm. The data obtained from four control and four L7-VGAT mice were averaged.VGAT transcripts , VGAT cRNA probes were synthesized using a digoxigenin (DIG) RNA Labeling Kit  and T3 RNA polymerase (Roche Diagnostics). To detect murine calbindin (CB) transcripts , CB cRNA probes were synthesized using a FITC-dUTP labeling kit (Roche Diagnostics) and Sp6 RNA polymerase (Roche Diagnostics) according to the manufacturer’s instructions.To detect murine VGAT and CB transcripts signals were obtained using a fluorescent microscope .The whole brains of mice at P2W, P4W, P8W, P16W, and P40W were removed and embedded in Tissue-Tek OCT compound. Samples were sectioned on a cryostat at 10 μm. The cryosections were mounted onto SuperFrost Plus microscope slides, air-dried for more than 20 min at room temperature, and stored at -80°C until use. The cryosections were fixed with 4% paraformaldehyde (PFA) for 10 min, and serial sections were treated according to previously described protocols , 2010. Tin situ hybridization (ISH) signals for both VGAT and CB. The percentage of VGAT mRNA-positive PCs was determined in lobule III, because lobule III was uniformly stained with our ISH protocol. We analyzed two sections per animal and used two animals per genotype at 2, 4, 8, 16, and 40 weeks of age . The number of VGAT mRNA-positive PCs was divided by the total number of PCs. Because CB is a marker of PCs . The composite images were prepared from the digital data files using Adobe Photoshop and Illustrator.To detect VGAT, CB, and PV, mice at P40W were transcardially perfused with 4% PFA in 0.1 M phosphate buffer, pH 7.4 (PB), under deep anesthesia with diethyl ether. Brains were harvested from the perfused animals, post-fixed in the identical fixative for 12 h at 4°C, and immersed in 30% sucrose in 0.1 M PB and 0.05% NaNprotocol . Alexa56ARα1 and 13% SDS–PAGE to detect GAT3, β-actin, and parvalbumin. Subsequently, the proteins were transferred to nitrocellulose membranes using a semidry transfer method. The membranes were reacted with rabbit anti-VGAT , and 1 mM phenylmethylsulfonyl fluoride (PMSF)). Homogenates were centrifuged at 3,000 rpm for 10 min at 4°C to obtain the supernatant as a crude fraction. The protein concentrations of the crude fractions were determined using the BCA protein assay reagent  and BSA as a standard. Equal amounts of protein from the crude fractions of the DCNs or cerebellar cortices were separated using 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) to detect VGAT, calbindin, gephyrin, and GABAT 1/100; , mouse a1 1/100; , rabbit The rotating rod apparatus  was used to evaluate the motor skill abilities of the mice. Male mice at postnatal 8, 16, and 40 weeks were tested with three trials per day for three consecutive days. Each trial lasted a maximum of 5 min during which time the rotating rod linearly underwent accelerated from 4 to 40 rpm. Animals were scored for their latency to fall (in seconds) in each trial. The animals were allowed at least 30 min of rest between trials to avoid fatigue and exhaustion. Statistical analyses were performed using two-way or one-way repeated measures ANOVA with the factors of genotype and trial.t-test.The footprint test was performed to examine the gaits of the mice. The rear paws of the mice were inked, and the mice were allowed to walk along a 50-cm-long, 10-cm-wide runway . A fresh sheet of paper was placed on the floor of the runway for each run. Each mouse was tested twice at P16W and P40W. The footprint patterns of the hind paws were evaluated in terms of the following two parameters (both measured in centimeters): (1) stride length, which was taken as the average distance between each consecutive pair of footprints for each hind paw; and (2) stride width, which was taken as the average perpendicular distance between each footprint and the line connecting each pair of consecutive footprints on the opposite side. Statistical analysis was performed using Student’s VGAT gene are flanked by loxP sequences . We crossed VGATflox/flox; L7+/+ mice with VGATflox/flox; L7Cre/+ mice to obtain VGATflox/flox; L7+/+ and VGATflox/flox; L7Cre/+ mice . We refer to VGATflox/flox; L7+/+ and VGATflox/flox; L7Cre/+ as the control and L7-VGAT mice, respectively, hereafter. In the L7-Cre knock-in mice, Cre recombinase is specifically expressed in the cerebellar PCs and retinal cells including the bipolar neurons and photoreceptor cells  . CB was used as a marker of PCs because CB is specifically expressed in PCs in the cerebellar cortex and DCNs . In contrast, no cells containing CB mRNA expressed VGAT mRNA in the L7-VGAT cerebellum at P40W . However, other VGAT mRNA signals were found in the molecular and granule cell layers of the P40W L7-VGAT mice. Furthermore, VGAT mRNA signals in brain regions outside of the cerebellum did not appear to be different between coronal sections of L7-VGAT and control mice at either P16W or P40W (data not shown).To assess whether and DCNs . In the VGAT mRNA was lost in the L7-VGAT PCs during development, we performed double ISH for VGAT and CB and counted the number of VGAT-positive PCs relative to the total number of PCs at five postnatal time points . The percentage of VGAT-positive PCs relative to the total PCs gradually decreased with age from 56% at P2W to 0% at P40W .Next, to assess the time at which Figure 3). CB is a PC marker protein that is located in the soma, dendrites, axonal fibers, and axon terminals of PCs . VGAT-immunoreactive signals overlapped with CB signals in the DCNs of the control mice, which suggests that the VGAT signals were derived from PCs. The DCN neurons appeared to be heavily innervated by GABAergic terminals of the PCs . In the L7-VGAT DCNs at P40W, the VGAT protein signals that were positive for CB had nearly disappeared . In contrast, the VGAT signals in the cerebellar cortices of both genotypes were similar . These results demonstrate that our L7-VGAT mice exhibited cerebellar PC-specific VGAT knockout at P40W. In the L7-VGAT DCNs at P40W, a small number of VGAT-positive and CB-negative signals remained. These results are consistent with a previous report that a large portion of inhibitory synapses on DCN neurons is derived from PCs . To acquire DCN tissue for Western blot analyses, we focused on GAT3. GAT3 is one of the GABA transporters, which uptake extracellular GABA, and is located in the glial cell membrane . We did not detect any overt abnormalities in the lamination or foliation of the 40-week-old L7-VGAT cerebellum . The sizes, densities, and staining intensities of the cells in each layer of the L7-VGAT cerebellar cortex were similar to those in control cerebellum . The overall size of DCN appeared to remain unaltered . Additionally, there were no significant differences in the thicknesses of the molecular or granule cell layers or in the densities of the PCs between the L7-VGAT and control cerebellar cortices (Table 2). These results suggest that the loss of VGAT in the PCs did not significantly interfere with the structure of the cerebellum.It is known that GABA can be involved in neural development . Thus, iFigures 3 and 6). CB is a presynaptic protein at the PC-DCN synapse. CB immunoreactivity, including punctate signals around the somata, was largely unaltered in the L7-VGAT DCNs . Moreover, PV, which is expressed in some neurons in the DCNs, was also localized to the PC terminals. The PV immunoreactive profiles were also similar between the control and L7-VGAT DCNs . As representatives of postsynaptic proteins, we examined the immunoreactivities for the GABA-A receptor α1 subunit  and gephyrin, which is a scaffolding protein for the GABAAR and glycine receptor . Additionally, the expression patterns of GABAARα1 and gephyrin in the L7-VGAT DCNs were similar to those in the control DCNs . We also performed Western blot analyses for CB, PV, GABAARα1, and gephyrin proteins in the crude fractions of the DCN to compare the expression levels of these proteins between the L7-VGAT and control mice . The amounts of these four proteins were not significantly different between the genotypes. Collectively, the distribution patterns and expression levels of the representative pre- and postsynaptic proteins were not different between the L7-VGAT and control mice.Next, we examined the immunohistochemical profiles of pre- and postsynaptic proteins at the PC-DCN synapse of the L7-VGAT cerebellum as an index of synapse formation . The control mice improved their performances with trial repetition, and even the older mice (P40W) demonstrated an improvement after repeated trials. At P8W, no significant differences were observed in the retention time between the L7-VGAT and control mice (p > 0.05), which indicates that the 8-week-old L7-VGAT mice performed normally in the accelerating rotarod test. At P16W, there was a significant difference in the retention time between the L7-VGAT and control mice (p < 0.001). However, there was no significant difference in the strain X trial interaction (p > 0.05). At P40W, there was a significant difference in the retention time between the L7-VGAT and control mice (p < 0.0001). Moreover, there was also a significant strain X trial interaction (p < 0.0001), which demonstrates that the L7-VGAT mice displayed severer ataxia at P40W than at P16W. However, the 40-week-old L7-VGAT mice began to exhibit moderately improved performance over trials (p < 0.0001).Because the cerebellum is important for motor coordination, we performed accelerating rotarod tests at P8W, P16W, and P40W . There were no significant differences in either stride length or width between the L7-VGAT and control mice at P16W . In contrast, at P40W, the stride lengths were similar, but the stride width of the L7-VGAT mice was significantly wider than that of the control mice  . These results support the conclusion that the L7-VGAT mice walked normally at P16W, but displayed abnormal ambulation at P40W.Next, we examined the footprint test . Moreover, clear alterations in the expression profiles of synaptic proteins, including CB, which is a marker protein for PCs in the cerebellum, were not observed , which suggests that there were no drastic disturbances in the PC-DCN synapse including in the axon terminals of the PCs. It has been reported that there are few structural abnormalities in mice with KOs of GABAergic system-related molecules including GAD and VGAT . Therefore, it is possible that developmental compensation occurred in the L7-VGAT cerebellum. On the other hand, there are reports that suggest that neurotransmitters that exert developmental effects are released in a non-vesicular fashion . Moreover, these impairments became more obvious as the deficit in VGAT progressed, which indicates that VGAT expression in cerebellar PCs plays an important role in the motor coordination of animals.L7-VGAT mice exhibited reduced retention times and widened gaits in the accelerating rotarod and footprint tests, respectively . However, the L7-VGAT mice displayed ataxia, which suggests that vesicularly released GABA from the cerebellar PC terminal greatly contributed to PC function.The cerebellum plays an important role in motor function, and PCs are indispensable for cerebellar function. The importance of PCs has been demonstrated by analyses of spontaneous mouse mutants in which cerebellar PCs are degenerated . HoweverVGAT mRNA-positive PCs remained, but widened gait was observed at P40W when all of the cells had disappeared . Immunoreactive signals for both CB and VGAT in the L7-VGAT DCNs at P16W were hardly detectable as they were at P40W (data not shown). These results suggest that the VGAT loss in the L7-VGAT PCs occurred at both the mRNA and protein levels during the same period. This assertion further indicates that gait disturbance can be prevented by the presence of only 5% of the normal population of vesicular GABA-releasing PCs. This inference is not compatible with the report that ataxia appears in the spontaneously PC-degenerating mutants before the number of surviving PCs is reduced by half , those in the DCN and VN of the spontaneously PC-degenerating mutants were altered. The number of puncta that were immunoreactive for gephyrin was reduced by approximately half . In the L7-VGAT DCN, the loss of GABAergic inhibition from PCs is expected. Nevertheless, the elevated expression of PV did not occur, which implies that the reorganization of the somatic motor pathway that occurs in the L7-VGAT CNS differs from that in the spontaneous mutants.PV is a Cae neuron . Althouge neuron . Moreoveant mice . MoreoveThe L7-VGAT mice generated in this study became completely PC-specific VGAT knockout mice and exhibited ataxia. It was expected that only vesicular release of GABA from the PCs would be disrupted in the L7-VGAT cerebellum and that PC degeneration and other drastic changes in cerebellar structure would not occur. The majority of mouse models of ataxia display histological phenomena, such as losses of PCs, which explain their cerebellar dysfunctions. Only a few mouse models exist that display ataxic behavior combined with normal cerebellar morphology . AdditioFigures 2 and 3). Because the L7-Cre gene in the L7-VGAT mice was heterozygous, the VGAT-flox gene would be expected to be deleted much earlier in the animals that are homozygous for the L7-Cre gene. The use of these two lines of L7-VGAT mice may be useful in the study of the differences in the effect of VGAT deficits that occur at different ages.The VGAT deficit was complete in the PCs at P40W (ARα1, and gephyrin, but that it was crucial for the motor coordination in animals. The L7-VGAT mice will be a useful model to provide a clue to the better understanding of PC function in diverse cerebellum-related behaviors.Studies using L7-VGAT mice demonstrated that VGAT in cerebellar PCs was not essential for the maintenance of overall cerebellar structure or the expression profiles of synaptic proteins including calbindin, parvalbumin, GABAMikiko Kayakabe, Toshikazu Kakizaki, Ryosuke Kaneko, Yoichi Nakazato, Koji Shibasaki, Yasuki Ishizaki, Nobuhiko Furuya, and Yuchio Yanagawa: Conceived and designed the experiments; Mikiko Kayakabe, Toshikazu Kakizaki, Ryosuke Kaneko, Atsushi Sasaki, Koji Shibasaki, and Yuchio Yanagawa: Performed the experiments; Mikiko Kayakabe, Toshikazu Kakizaki, Ryosuke Kaneko, Atsushi Sasaki, Koji Shibasaki, and Yuchio Yanagawa: Analyzed the data; Ryosuke Kaneko, Atsushi Sasaki, Yoichi Nakazato, Hiromitsu Saito, and Noboru Suzuki: Contributed new reagents/analytical tools; Mikiko Kayakabe, Toshikazu Kakizaki, Ryosuke Kaneko, and Yuchio Yanagawa:Wrote the paper. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A single-step blending approach allows genomic prediction using information ofgenotyped and non-genotyped animals simultaneously. However, the combinedrelationship matrix in a single-step method may need to be adjusted becausemarker-based and pedigree-based relationship matrices may not be on the samescale. The same may apply when a GBLUP model includes both genomic breeding valuesand residual polygenic effects. The objective of this study was to comparesingle-step blending methods and GBLUP methods with and without adjustment of thegenomic relationship matrix for genomic prediction of 16 traits in the NordicHolstein population.The data consisted of de-regressed proofs (DRP) for 5 214 genotyped and 9 374non-genotyped bulls. The bulls were divided into a training and a validationpopulation by birth date, October 1, 2001. Five approaches for genomic predictionwere used: 1) a simple GBLUP method, 2) a GBLUP method with a polygenic effect, 3)an adjusted GBLUP method with a polygenic effect, 4) a single-step blendingmethod, and 5) an adjusted single-step blending method. In the adjusted GBLUP andsingle-step methods, the genomic relationship matrix was adjusted for thedifference of scale between the genomic and the pedigree relationship matrices. Aset of weights on the pedigree relationship matrix (ranging from 0.05 to 0.40) wasused to build the combined relationship matrix in the single-step blending methodand the GBLUP method with a polygenetic effect.Averaged over the 16 traits, reliabilities of genomic breeding values predictedusing the GBLUP method with a polygenic effect (relative weight of 0.20) were 0.3%higher than reliabilities from the simple GBLUP method (without a polygeniceffect). The adjusted single-step blending and original single-step blendingmethods (relative weight of 0.20) had average reliabilities that were 2.1% and1.8% higher than the simple GBLUP method, respectively. In addition, the GBLUPmethod with a polygenic effect led to less bias of genomic predictions than thesimple GBLUP method, and both single-step blending methods yielded less bias ofpredictions than all GBLUP methods.The single-step blending method is an appealing approach for practical genomicprediction in dairy cattle. Genomic prediction from the single-step blendingmethod can be improved by adjusting the scale of the genomic relationshipmatrix. Selection based on dense markers across the genome  has becoMany studies have shown that a linear model which assumes that effects of all singlenucleotide polymorphisms (SNP) are normally distributed with equal variance performs aswell as variable selection models for most traits in dairy cattle ,4. BecauSeveral blending strategies, including multi-step and single-step approaches, have beenproposed to estimate GEBV ,5,12-18.Some studies -15,18 haThe purpose of this study was to compare single-step blending and GBLUP methods with andwithout adjustment of the genomic relationship matrix for genomic prediction of 16traits in the Nordic Holstein population. De-regressed proofs were used as responsevariables in both GBLUP and the single-step blending methods.Data consisted of 5 214 genotyped bulls born between 1974 and 2008 and 9 374non-genotyped bulls born between 1950 and 2008. The bulls were divided into atraining and a validation population by birth date, October 1, 2001. Thus, thetraining data contained 3 045 genotyped and 8 822 non-genotyped bulls born beforethis date, and the validation data contained 2 169 genotyped bulls born after thisdate. Non-genotyped bulls born after October 1, 2001 were not used in training orvalidation. For the GBLUP methods described below, the training data only includedthe 3 045 genotyped animals. All 16 traits (sub-indices) in the Nordic Total Meritindex were assessed, including yield, conformation, fertility, and health traits. Foreach trait, the DRP with reliability less than 0.20 were excluded from the trainingand the validation data. This removed 1.3%, 2.8% and 3.2% of DRP for birth index,fertility and health, respectively, and less than 0.5% for the other traits. Thenumbers of individuals in the training and validation datasets differed betweentraits . The final marker data included 48 073 SNPs for 5 214 bulls afterremoving SNP with minor allele frequency (MAF) less than 0.01 and locus averageGenCall score less than 0.60.http://www.nordicebv.info/Routine+evaluation/.De-regressed proofs (DRP) were used as response variables for genomic prediction inall approaches. Based on EBV data of 14 588 progeny-tested bulls and pedigree data of42 144 animals, the de-regression was carried out by applying the iterative proceduredescribed in ,24 usingThree GBLUP and two single-step blending methods were used. All analyses wereperformed with the DMU package ,27, for The basic GBLUP method ,29 used y is the data vector of DRP of genotyped bulls, μ is the overallmean, 1 is a vector of ones, Z is a design matrix that allocatesrecords to breeding values, g is a vector of DGV to be estimated, ande is a vector of residuals. It was assumed that G isthe marker-based genomic relationship matrix [G were estimated from theobserved genotype data. Random residuals were assumed such thatD is a diagonalmatrix with elements i account for heterogeneousresidual variances due to differences in reliabilities of DRP. They were definedas where p matrix ,29. Alleu is the vector of residual polygenic effects that are not capturedby the SNP.where A is the pedigree-based relationshipmatrix. Define Here, we used an equivalent approach. Let Gω is a combinedrelationship matrix, gω weredefined as DGVω to distinguish from the simple GBLUP and thesingle-step blending methods.It was assumed that G was adjusted to be on the same scale as A. Then, the combinedrelationship matrix was G* is the adjusted genomicrelationship matrix. The adjustment of G is described below.The model was the same as the above GBLUP method with a polygenic effect butG with thepedigree-based numerator relationship matrix A, using the followingmodel:The original single-step blending method ,17,18 usy is the vector of DRP for both genotyped and non-genotyped bulls,1 is a vector of ones, Z is a design matrix, and a is thevector of additive genetic effects, which are the sum of the genomic and theresidual polygenic effects. It was assumed that H is the modified geneticrelationship matrix that combines pedigree-based relationship information [where ormation ,15:A11 is the sub-matrix of the pedigree-based relationshipmatrix (A) for genotyped animals, A22 is the sub-matrixof A for non-genotyped animals, A12 (orA21) is the sub-matrix of A for relationships betweengenotyped and non-genotyped animals, and G matrix used in the single-step blendingwas the same as in the GBLUP method. The inverse of H[where erse of H,17 isG matrix was adjusted forthe difference between the original genomic relationship matrix and pedigreerelationship matrix (A11), as proposed by previous studies [G matrix was adjusted using two parameters α andβ [In the adjusted single-step blending method, the  studies ,20. The  α andβ , i.e.,which were derived from the following equations:G* was then used to replace G to construct thecombined relationship matrix in the single-step blending method.Matrix Gω and The weights ω ranging from 0.05 to 0.40 were used to constructThe reliabilities of genomic predictions were measured as squared correlationsbetween the predicted breeding values and DRP for bulls in the validation data,divided by the average reliability of the DRP in validation data. AHotelling-Williams t-test was used to test the difference between the validationcorrelations obtained from these five prediction methods ,31. BiasGenomic predictions using the GBLUP method were improved when a polygenic effect wasincluded Tables  and 3. Wω from the GBLUP method with apolygenic effect (the adjusted GBLUP method is shown as an example) for the 16 traits,with a relative weight ω = 0.20. The adjusted single-step blending ledto the highest reliability of genomic predictions, followed by the original single-stepblending, and the GBLUP method resulted in the lowest reliability. Reliabilities rangedfrom 0.206 to 0.503 (average 0.379) for the original single-step blending, from 0.206 to0.503 (average 0.382) for the adjusted single-step blending, and from 0.183 to 0.481(average 0.363) for the GBLUP method. In general, single-step blending was better thanthe GBLUP method and adjusted single-step blending was better than the originalsingle-step blending, especially for production traits. On average, reliabilities ofgenomic breeding values predicted using the original single-step blending were 1.6 %higher than reliabilities from the adjusted GBLUP method, but 0.3% lower thanreliabilities from the adjusted single-step blending.Table The regression coefficients Table  ranged fTable Gω and H, eight values of ω between 0.05 and 0.40were used for the two single-step blending methods and the two GBLUP methods with apolygenic effect. On average, reliabilities varied from 0.356 to 0.363 over the eightscenarios for the two GBLUP methods, from 0.372 to 0.379 for the original single-stepblending, and from 0.374 to 0.382 for the adjusted single-step blending , while a coefficientlarger than 1 indicates underestimation (deflation). The two single-step blendingmethods led to less bias than the three GBLUP methods, and the two GBLUP methods with apolygenic effect resulted in less bias than the simple GBLUP method without a polygeniceffect. The problem of inflation of genomic predictions is critical in practice -35 as itu et al.  found thG matrix that is based on current allele frequencies and suggested that theoptimal G has average of diagonal and off-diagonal elements close to those ofA11. Forni et al. [G matrix is a reasonable solution toavoid inflation in pig data. However, in the present study, the adjusted G matrixdid not improve genomic predictions in the GBLUP methods with a polygenic effect. Thissuggests that, based on the present data, adjustment of G has little effect ongenomic prediction when only genotyped animals are used, but may be important in otherdata where there is a large difference in scale between G and A.The adjusted single-step blending method resulted in less bias than the originalsingle-step blending for all settings of the weight factor. In a simulation study,Vitezica et al.  also foui et al.  also shoThe results from the present study indicate that increasing the weighting factor (0.40)reduces bias and that weighting factors around 0.15 to 0.20 give the highest reliabilitybut the optimal weighting factors differed between traits. Similarly, Liu et al.  observedGmatrix is much larger, compared with the results from the current study. This may bebecause there was more inbreeding in the pig data, which resulted in average values ofthe diagonal and off-diagonal elements of A11 equal to 1.145 and0.298, and estimates of β and α equal to 0.895 and 0.298, respectively. In thepresent study, the averages of the diagonal and off-diagonal elements ofA11were 1.060 and 0.085, and estimates of β and α were0.976 and 0.085, i.e. closer to one and zero, respectively. This means that the originalG matrix was less adjusted in this study compared to the study on pig data byChristensen et al. [Christensen et al.  comparedn et al. .The single-step blending methods can increase reliability and reduce bias of genomicpredictions. The adjusted single-step blending method performed slightly better than theoriginal single-step blending method, both with respect to reliability and bias ofgenomic predictions. The weighting factor used in these single-step blending methods hada small effect on reliability of genomic prediction but an important effect on bias.The authors declare that they have no competing interests.HG performed statistical analysis and wrote the manuscript. OFC derived the single-stepmethods and improved the manuscript. PM provided the software, helped to the analysisand added valuable comments. USN prepared the data. GS and MSL conceived the study, madesubstantial contribution for the results interpretation and revised the manuscript. MSL,GS and YZ coordinated the project. All authors read and approved the manuscript.


N-methyl-D-aspartate (NMDA) receptors are involved in the oxaliplatin-induced mechanical allodynia. In the present study, we investigated the involvement of Ca2+/calmodulin dependent protein kinase II (CaMKII), which is a major intracellular protein kinase and is activated by NMDA receptor-mediated Ca2+ influx, in the oxaliplatin-induced mechanical allodynia in rats.Oxaliplatin is a key drug in the treatment of colorectal cancer, but it causes severe peripheral neuropathy. We previously reported that oxaliplatin  induces mechanical allodynia in the late phase in rats, and that spinal NR2B-containig 4-6) of oxaliplatin-treated rats. This increased CaMKII phosphorylation was reversed by intrathecal injection of a selective CaMKII inhibitor KN-93  and a selective NR2B antagonist Ro 25-6981 . Moreover, acute administration of KN-93  strongly reversed the oxaliplatin-induced mechanical allodynia in von Frey test, while it did not affect the oxaliplatin-induced cold hyperalgesia in acetone test. Similarly, oral administration of trifluoperazine , which is an antipsychotic drug and inhibits calmodulin, reduced both mechanical allodynia and increased CaMKII phosphorylation. On the other hand, trifluoperazine at the effective dose (0.3 mg/kg) had no effect on the paw withdrawal threshold in intact rats. In addition, trifluoperazine at the same dose did not affect the motor coordination in rota-rod test in intact and oxaliplatin-treated rats.An increase of CaMKII phosphorylation was found in the spinal cord (LThese results suggest that CaMKII is involved in the oxaliplatin-induced mechanical allodynia, and trifluoperazine may be useful for the treatment of oxaliplatin-induced peripheral neuropathy in clinical setting. Oxaliplatin, a platinum-based chemotherapeutic agent, has widely been used for colorectal cancer. However, oxaliplatin causes severe peripheral neuropathy. After multiple cycles, the patients develop a chronic neuropathy that is characterized by a sensory and motor dysfunction. This chronic neuropathy is a dose-limiting toxicity and a major clinical problem in oxaliplatin-based chemotherapy .N-methyl-D-aspartate (NMDA) receptors are involved in the oxaliplatin-induced mechanical allodynia [We previously reported that repeated administration of oxaliplatin induced cold hyperalgesia in the early phase and mechanical allodynia in the late phase in rats . Recentlllodynia . The NMD2+ influx into the cytosol. This increased Ca2+ influx initiates cascades of intracellular signaling events involving Ca2+ and various protein kinases [2+/calmodulin dependent protein kinase II (CaMKII) is a major intracellular protein kinase and is activated by Ca2+ signaling [2+ initially activates calmodulin by binding to its Ca2+-binding sites, and this interaction induces a change in the conformation of calmodulin. CaMKII is then switched to an activated state by exposure to Ca2+/calmodulin. Several studies showed that an increase of CaMKII activation in the spinal cord is involved in persistent pain by nerve injury [Activation of the NMDA receptors leads to an increase in Ca kinases . Ca2+/caignaling . An incre injury -9 and ine injury ,11. Howep < 0.01, Figure p < 0.05, Figure Oxaliplatin  significantly reduced the paw withdrawal thresholds compared with the vehicle in the von Frey test on day 24  significantly increased the number of withdrawal responses to cold stimulation by acetone spray on day 5 . The pCaMKII in the spinal cord of oxaliplatin treated rats significantly increased compared with that of vehicle-treated rats on day 25  and 120 min (0.3 mg/kg: p < 0.05) after the administration  and 30 min after administration of trifluoperazine, there was no difference in the motor coordination among the groups-treated with distilled water, trifluoperazine 0.3 mg/kg or oxaliplatin + trifluoperazine 0.3 mg/kg Figure .2+ channels [2+ channels. However, its structural analog KN-92, which does not inhibit CaMKII but blocks L-type Ca2+ channels' current, did not reverse the oxaliplatin-induced mechanical allodynia. Moreover, the expression of pCaMKII significantly increased in the spinal cord of oxaliplatin-treated rats on day 25, and this increase of pCaMKII was blocked by KN-93. On the other hand, KN-93 had no effect on the oxaliplatin-induced cold hyperalgesia on day 5. These results indicate that the spinal CaMKII is involved in the oxaliplatin-induced mechanical allodynia but not cold hyperalgesia.In the present study, acute treatment of the CaMKII inhibitor KN-93 reversed the oxaliplatin-induced mechanical allodynia. KN-93 binds directly to calmodulin-binding site of CaMKII and inhibits the enzyme activity . KN-93 achannels , and the2+ influx through activated NMDA receptors causes multiple intracellular changes including an activation of CaMKII [2+ concentration by glutamate stimulation, leads to the reduction of spinal CaMKII phosphorylation in the neuropathic pain model [Recently, we reported that repeated administration of oxaliplatin increased the expression of NR2B protein and mRNA in the rat spinal cord on day 25 (late phase) but not on day 5 (early phase), and Ro 25-6981 reversed the oxaliplatin-induced mechanical allodynia , suggestf CaMKII ,19, and f CaMKII . FurtherIn this study, we confirmed an inhibition of CaMKII phosphorylation by trifluoperazine, which inhibits calmodulin required for CaMKII activation . FurtherOur results indicate that repeated administration of oxaliplatin increases spinal CaMKII activity. This increase of CaMKII activation was reversed by intrathecal injection of the selective CaMKII inhibitor and the selective NR2B antagonist. This CaMKII activation may contribute to the incidence of mechanical allodynia. Furthermore, the selective CaMKII inhibitor and the selective NR2B antagonist reduced the oxaliplatin-induced pain behavior. In addition, trifluoperazine reduced the oxaliplatin-induced mechanical allodynia and CaMKII activation. These results suggest that inhibition of CaMKII or NMDA-CaMKII pathway provides a novel therapeutic target for the treatment of the oxaliplatin-induced peripheral neuropathy.Male Sprague-Dawley rats weighing 200-250 g  were used in the present study. Animals were housed in groups of four to five per cage, with lights on from 7:00 to 19:00 h. Animals had free access to food and water in their home cages. All experiments were approved by the Experimental Animal Care and Use Committee of Kyushu University according to the National Institutes of Health guidelines, and we followed International Association for the Study of Pain (IASP) Committee for Research and Ethical Issues guidelines for animal research .Oxaliplatin (Elplat®) was obtained from Yakult Co., Ltd. . KN-93, Ro 25-6981 hydrochloride hydrate and trifluoperazine dihydrochloride were purchased from Sigma-Aldrich . KN-92 was purchased from Calbiochem . Oxaliplatin was dissolved in 5% glucose solution. The vehicle-treated rats were injected with 5% glucose solution. KN-93, KN-92 and Ro 25-6981 were dissolved in 100% dimethyl sulfoxide (DMSO). Trifluoperazine was dissolved in distilled water. The doses of these drugs were chosen based on previous reports .Mechanical allodynia and cold hyperalgesia were induced according to the method described previously . OxaliplBehavioral test was performed blindly with respect to drug administration.The mechanical allodynia was assessed by von Frey test. Each rat was placed in a clear plastic box (20 × 17 × 13 cm) with a wire mesh floor and allowed to habituate for 30 min prior to testing. von Frey filaments  ranging from 1- to 15-g bending force were applied to the midplantar skin of each hind paw six times, with each application held for 6 s. The paw withdrawal threshold was determined by a modified up-down method .The cold hyperalgesia was assessed by acetone test. Each rat was placed in a clear plastic box (20 × 17 × 13 cm) with a wire mesh floor and allowed to habituate for 30 min prior to testing. Fifty microliters of acetone  was sprayed onto the plantar skin of each hind paw 3 times, and the number of withdrawal responses was counted for 40 s from the start of the acetone spray.The rota-rod test was performed to investigate the change of motor coordination. Rats were placed on a rotating rod  and the latency to falling was measured for up to 2 min according to the method described previously . The tesWe confirmed the incidence of mechanical allodynia in the von Frey test on day 24. We carried out the drug evaluation on the next day. KN-93 (10-50 nmol) or KN-92 (50 nmol) was administered i.t. injection by direct lumbar puncture in a volume of 50 µL. The von Frey test was performed immediately before (0 min) and at 30, 60, 90 and 120 min after administration of the drugs. Trifluoperazine (0.05-0.3 mg/kg) was administered p.o. The von Frey test was performed immediately before (0 min) and at 30, 120 and 240 min after oral administration of trifluoperazine.We confirmed the incidence of cold hyperalgesia in the acetone test on day 5. KN-93 (10-50 nmol) was administered i.t. injection by direct lumbar puncture in a volume of 50 µL. The acetone test was performed immediately before (0 min) and at 30, 60, 90 and 120 min after administration of the drug.We investigated the effect of trifluoperazine on the mechanical nociceptive threshold in the von Frey test. Trifluoperazine (0.3 mg/kg) was administered p.o. in intact rats. The von Frey test was performed immediately before (0 min) and at 30, 120 and 240 min after oral administration of trifluoperazine.We investigated the effect of trifluoperazine on the motor coordination in the rota-rod test. Trifluoperazine (0.3 mg/kg) was administered p.o. in intact and oxaliplatin-treated rats. The rota-rod test was performed immediately before (0 min) and at 30 min after oral administration of trifluoperazine.4-6) of the spinal cord were quickly removed at 30 min after administration of KN-93 , Ro 25-6981  or trifluoperazine  on day 25. The tissues were homogenized in a solubilization buffer containing 20 mM Tris-HCl , and the homogenates were subjected to 12.5% SDS-PAGE, and proteins were transferred electrophoretically to PVDF membranes. The membranes were blocked in Tris-buffered saline Tween-20 (TBST) containing 5% BSA (Sigma-Aldrich) for an additional 1 h at room temperature with agitation. The membrane was incubated overnight at 4°C with mouse polyclonal anti-CaMKIIα antibody or rabbit polyclonal anti-(Thr286)pCaMKII  and then incubated for 1 h with corresponding horseradish peroxidase conjugate secondary antibodies . The immunoreactivity was detected using Enhanced Chemiluminescence . Ratios of the optical densities of pCaMKII to those of CaMKII were calculated for each sample.The lumbar sections  followed by the Tukey-Kramer post-hoc test  to determine differences among the groups. A p value of less than 0.05 is considered as statistically significant.Values were expressed as the means ± SEM. The values were analyzed by the Student's The authors declare that they have no competing interests.NE and TK are responsible for experimental design. MS, SU and HS are responsible for performance of behavioral tests. MS, SU, SY, HS and KM are responsible for performance of Western blotting. NE, MS and RO are responsible for writing the manuscript. All authors read and approved the final manuscript.